Single-cell RNA sequencing identifies critical transcription factors of tumor cell invasion induced by hypoxia microenvironment in glioblastoma.

Rationale: Glioblastoma (GBM) is an aggressive malignant primary brain cancer with poor survival. Hypoxia is a hallmark of GBM, which promotes tumor cells spreading (invasion) into the healthy brain tissue. Methods: To better elucidate the influence of hypoxia on GBM invasion, we proposed a data-driven modeling framework for predicting cellular hypoxia (CHPF) by integrating single cell transcriptome profiling and hypoxia gene signatures. Results: We characterized the hypoxia status landscape of GBM cells and observed that hypoxic cells were only present in the tumor core. Then, by investigating the cell-cell communication between immune cells and tumor cells, we discovered significant interaction between macrophages and tumor cells in hypoxic microenvironment. Notably, we dissected the functional heterogeneity of tumor cells and identified a hypoxic subpopulation that had highly invasive potential. By constructing cell status specific gene regulatory networks, we further identified 14 critical regulators of tumor invasion induced by hypoxic microenvironment. Finally, we confirmed that knocking down two critical regulators CEBPD and FOSL1 could reduce the invasive ability of GBM under hypoxic conditions. Additionally, we revealed the therapeutic effect of Axitinib and Entinostat through the mice model. Conclusion: Our work revealed the critical regulators in hypoxic subpopulation with high invasive potential in GBM, which may have practical implications for clinical targeted-hypoxia cancer drug therapy.

Characterization of the metabolic alteration-modulated tumor microenvironment mediated by TP53 mutation and hypoxia.

BACKGROUND: TP53 mutation and hypoxia play an essential role in cancer progression. However, the metabolic reprogramming and tumor microenvironment (TME) heterogeneity mediated by them are still not fully understood. METHODS: The multi-omics data of 32 cancer types and immunotherapy cohorts were acquired to comprehensively characterize the metabolic reprogramming pattern and the TME across cancer types and explore immunotherapy candidates. An assessment model for metabolic reprogramming was established by integration of multiple machine learning methods, including lasso regression, neural network, elastic network, and survival support vector machine (SVM). Pharmacogenomics analysis and in vitro assay were conducted to identify potential therapeutic drugs. RESULTS: First, we identified metabolic subtype A (hypoxia-TP53 mutation subtype) and metabolic subtype B (non-hypoxia-TP53 wildtype subtype) in hepatocellular carcinoma (HCC) and showed that metabolic subtype A had an "immune inflamed" microenvironment. Next, we established an assessment model for metabolic reprogramming, which was more effective compared to the traditional prognostic indicators. Then, we identified a potential targeting drug, teniposide. Finally, we performed the pan-cancer analysis to illustrate the role of metabolic reprogramming in cancer and found that the metabolic alteration (MA) score was positively correlated with tumor mutational burden (TMB), neoantigen load, and homologous recombination deficiency (HRD) across cancer types. Meanwhile, we demonstrated that metabolic reprogramming mediated a potential immunotherapy-sensitive microenvironment in bladder cancer and validated it in an immunotherapy cohort. CONCLUSION: Metabolic alteration mediated by hypoxia and TP53 mutation is associated with TME modulation and tumor progression across cancer types. In this study, we analyzed the role of metabolic alteration in cancer and propose a predictive model for cancer prognosis and immunotherapy responsiveness. We also explored a potential therapeutic drug, teniposide.

A preliminary study of further attempt at the development, testing and application of an independent primary screening stool card.

Stool characteristics are of great value to assess diseases, but patients knew little. E-learning applied in health popularization and patient education is booming. In China, WeChat applets has advantages of abundant users, convenient access and low cost, which may be a great media in patient education on stool. This preliminary study aims to develop and evaluate a stool card WeChat applet. We collected stools images during 2020 to 2022 in the Department of Gastroenterology and Hepatology in the Second Affiliated Hospital of Harbin Medical University, constructed a stool card applet named the Doctor Friend Primary Screening Stool Card (DFPSSC) and evaluated it. Eligible participants were divided into the applet, traditional paper media and control group. We implement a series of tests to evaluate the effectiveness. 20 clinicians and participants using the DFPSSC completed a questionnaire to evaluate the usability. We developed the DFPSSC for an E-learning approach. Of 108 volunteers, 97 completed the DFPSSC learning. No significant pretest differences were found among the three groups (P = 0.303). Applet group had significantly higher posttest scores than pretest scores in intervention (P < 0.001, d = 1.68) and simulation (P = 0.006) test, and it had higher scores than other two group (P < 0.001). 63% participants and 59% clinicians strongly agree or agree to the usability of DFSSC. This preliminary study verified that the DFPSSC can effectively improve participants' knowledge of feces, making it an effective clinical tool for patient education and the avoidance of treatment delay.

From Hair to Colon: Hair Follicle-Derived MSCs Alleviate Pyroptosis in DSS-Induced Ulcerative Colitis by Releasing Exosomes in a Paracrine Manner.

Ulcerative colitis (UC) has attracted intense attention due to its high recurrence rate and the difficulty of treatment. Pyroptosis has been suggested to be crucial in the development of UC. Although mesenchymal stem cells (MSCs) are broadly used for UC therapy, they have rarely been studied in the context of UC pyroptosis. Hair follicle-derived MSCs (HFMSCs) are especially understudied with regard to UC and pyroptosis. In this study, we aimed to discover the effects and potential mechanisms of HFMSCs in UC. We administered HFMSCs to dextran sulfate sodium- (DSS-) treated mice and found that the HFMSCs significantly inhibited pyroptosis to alleviate DSS-induced UC. A transwell system and GW4869, an exosome inhibitor, were used to prove the paracrine mechanism of HFMSCs. HFMSC supernatant reduced pyroptosis-related protein expression and promoted cell viability, but these effects were attenuated by GW4869, suggesting a role for HFMSC-released exosomes (Exos) in pyroptosis. Next, Exos were extracted and administered in vitro and in vivo to explore their roles in pyroptosis and UC. In addition, the biodistribution of Exos in mice was tracked using an imaging system and immunofluorescence. The results suggested that Exos not only improved DSS-induced pyroptosis and UC but also were internalized into the injured colon. Furthermore, the therapeutic efficacy of Exos was dose dependent. Among the Exo treatments, administration of 400 µg of Exos per mouse twice a week exhibited the highest efficacy. The differentially expressed miRNAs (DEmiRNAs) between MSCs and MSC-released Exos suggested that Exos might inhibit pyroptosis through tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) signalling and interferon- (IFN-) gamma pathways. Our study reveals that HFMSCs can alleviate pyroptosis in UC by releasing DEmiRNA-containing Exos in a paracrine manner. This finding may lead to new treatments for UC.

Fucosyltransferase 4 Predicts Patient Outcome in Rectal Cancer through an Immune Microenvironment-Mediated Multi-Mechanism.

Colorectal cancer is the most common type of gastrointestinal malignant tumors worldwide. Standardization of the strategy for the precise treatment of this cancer has been a major challenge. Enrichment analysis of six gene groups (colon cancer-specific genes (upregulated and downregulated); rectal cancer-specific genes (upregulated and downregulated); and common genes (upregulated and downregulated)) revealed the common and specific features of colon and rectal cancer, particularly a hyperactive immune response in rectal cancer. Key common genes exhibited a similar expression pattern, but were associated with distinct patient prognosis in colon and rectal cancer. FUT4 was a core regulatory gene in rectal cancer; it can decrease the level of infiltration by M2 macrophages in the tumor immune microenvironment and participate in the positive regulation of the immune system and glycoprotein biosynthetic process, thereby affecting the outcome of patients with rectal cancer. FUT4 co-expression genes can influence patient's survival time by regulating the cell cycle. Among the regulators of FUT4 co-expression genes, checkpoint kinase 2 (CHEK2) was linked to patient outcome.

From hair to liver: emerging application of hair follicle mesenchymal stem cell transplantation reverses liver cirrhosis by blocking the TGF-ß/Smad signaling pathway to inhibit pathological HSC activation.

Liver cirrhosis (LC) involves multiple systems throughout the body, and patients with LC often die of multiple organ failure. However, few drugs are useful to treat LC. Hair follicle mesenchymal stem cells (HF-MSCs) are derived from the dermal papilla and the bulge area of hair follicles and are pluripotent stem cells in the mesoderm with broad prospects in regenerative medicine. As an emerging seed cell type widely used in skin wound healing and plastic surgery, HF-MSCs show considerable prospects in the treatment of LC due to their proliferation and multidirectional differentiation capabilities. We established an LC model in C57BL/6J mice by administering carbon tetrachloride (CCl4) and injected HF-MSCs through the tail vein to explore the therapeutic effects and potential mechanisms of HF-MSCs on LC. Here, we found that HF-MSCs improved liver function and ameliorated the liver pathology of LC. Notably, PKH67-labeled HF-MSCs were detected in the injured liver and expressed the hepatocyte-specific markers cytokeratin 18 (CK18) and albumin (ALB). In addition, in contrast to that in the LC group, the α-SMA expression showed a decreasing trend in the treatment group in vitro and in vivo, indicating that the pathological activation of hepatic stellate cells (HSCs) was inhibited by HF-MSC treatment. Moreover, the levels of transforming growth factor ß (TGF-ß1) and p-Smad3, a signaling molecule downstream of TGF-ß1, were increased in mice with LC, while HF-MSC treatment reversed these changes in vivo and in vitro. Based on these findings, HF-MSCs may reverse LC by blocking the TGF-ß/Smad pathway and inhibiting the pathological activation of HSCs, which may provide evidence for the application of HF-MSCs to treat LC.

ECM1 modified HF-MSCs targeting HSC attenuate liver cirrhosis by inhibiting the TGF-ß/Smad signaling pathway.

Hair follicle-derived mesenchymal stem cells (HF-MSCs) show considerable therapeutic potential for liver cirrhosis (LC). To improve the effectiveness of naïve HF-MSC treatments on LC, we used bioinformatic tools to identify an exogenous gene targeting HSCs among the differentially expressed genes (DEGs) in LC to modify HF-MSCs. Extracellular matrix protein 1 (ECM1) was identified as a DEG that was significantly downregulated in the cirrhotic liver. Then, ECM1-overexpressing HF-MSCs (ECM1-HF-MSCs) were transplanted into mice with LC to explore the effectiveness and correlated mechanism of gene-overexpressing HF-MSCs on LC. The results showed that ECM1-HF-MSCs significantly improved liver function and liver pathological injury in LC after cell therapy relative to the other treatment groups. Moreover, we found that ECM1-HF-MSCs homed to the injured liver and expressed the hepatocyte-specific surface markers ALB, CK18, and AFP. In addition, hepatic stellate cell (HSC) activation was significantly inhibited in the cell treatment groups in vivo and in vitro, especially in the ECM1-HF-MSC group. Additionally, TGF-ß/Smad signal inhibition was the most significant in the ECM1-HF-MSC group in vivo and in vitro. The findings indicate that the genetic modification of HF-MSCs with bioinformatic tools may provide a broad perspective for precision treatment of LC.

Integrated Analysis of Angiogenesis-Mediated Tumor Immune Microenvironment Pattern in Hepatocellular Carcinoma (HCC) and a Novel Prognostic Model Construction to Predict Patient Outcome.

BACKGROUND Hepatocellular carcinoma (HCC) is a malignant tumor which is famous for its high heterogeneity and complex pathogenesis. Angiogenesis is an important driver of tumor progression and immune-suppressive microenvironment formation. MATERIAL AND METHODS A training set was acquired from the TCGA-LIHC cohort. An angiogenesis-active subtype was identified by consensus clustering analysis. The tumor subtype's immune microenvironment pattern was analyzed using quanTIseq. DEGs-mediated biology function was analyzed by enrichment analysis based on GO and KEGG. A prognostic model was constructed using LASSO Cox regression analysis and validated by 2 external datasets derived from GEO and ICGC. Quantitative real-time PCR assay was conducted to analyze CDCA8's expression status in the HCC line and normal liver cell line. RESULTS In HCC, patients with the angiogenesis-active subtype had a poor prognosis. Angiogenesis can shape the tumor microenvironment into high-M2 microphage infiltration and activity pattern. Here, we identified an angiogenesis-active HCC subtype and constructed an angiogenesis feature-based prognostic model to predict patient outcome. The external validation sets were enrolled to verify the accuracy of this model. CONCLUSIONS Our research demonstrated angiogenesis can confer the tumor immune-suppressive characteristic. We provide a robust method to evaluate the HCC's angiogenesis potential and help identify the angiogenesis-active subtype. Validation in the external validation cohort further confirmed the accuracy of our prognostic model.

TGF-ß1 signaling pathway serves a role in HepG2 cell regulation by affecting the protein expression of PCNA, gankyrin, p115, XIAP and survivin.

The transforming growth factor-ß (TGF-ß) signaling pathway serves a key role in the pathogenesis of liver cancer. To investigate the association between TGF-ß1 and the following proteins: Proliferating cell nuclear antigen (PCNA), gankyrin, general vesicular transport factor p115 (p115), X-linked inhibitor of apoptosis protein (XIAP) and survivin, HepG2 liver cancer cells were transfected with small interfering RNA (siRNA) directed against TGF-ß1, or were treated with exogenous TGF-ß1. TGF-ß1 protein expression levels were assessed at 72 and 96 h using western blotting, cell growth was evaluated using a Cell Counting kit-8 assay, and flow cytometry was used to examine cell cycle distribution and apoptosis. In addition, PCNA, gankyrin, p115, XIAP and survivin protein levels were evaluated using western blotting. TGF-ß1 protein expression levels were decreased at 72 and 96 h following siRNA transfection, indicating that the siRNA against TGF-ß1 was effective. In the TGF-ß1-knockdown group, the HepG2 cells exhibited G1 or S-phase cell cycle arrest; therefore, the number of G2-phase cells was decreased, cell growth was inhibited and apoptotic peaks were observed. By contrast, no significant alteration in cell cycle distribution or apoptosis was observed in the cells treated with exogenous TGF-ß1. In the exogenous TGF-ß1 group, PCNA and XIAP protein expression levels were increased, whereas gankyrin, p115 and survivin protein expression was observed to be dependent on the duration of treatment. By contrast, PCNA, gankyrin, XIAP and survivin protein expression decreased following TGF-ß1 knockdown; however, p115 protein expression increased. In conclusion, the TGF-ß1 signaling pathway may affect cell growth, cell cycle distribution and apoptosis through the regulation of PCNA, gankyrin, p115, XIAP and survivin protein expression in liver cancer. The results of the present study may improve the current understanding of the role of the TGF-ß signaling pathway during the pathogenesis of liver cancer.

Simultaneous silencing of ß-catenin and signal transducer and activator of transcription 3 synergistically induces apoptosis and inhibits cell proliferation in HepG2 liver cancer cells.

The tumorigenesis and maintenance of a cancer cells is dependent upon the collaboration of multiple signaling pathways. Signal transducer and activator of transcription 3 (STAT3) and ß-catenin are at the center of multiple cancer-associated signaling pathways; therefore, simultaneously targeting STAT3 and ß-catenin may be a potential cancer treatment, leading to induced lethality of cancer cells. In the present study, HepG2 liver cancer cells were transfected with small interfering RNA (siRNA) against ß-catenin and STAT3 alone or in combination. The cell growth was assessed using an MTT assay and the levels of cell apoptosis were detected using flow cytometry. Protein levels of caspase-3, cleaved caspase-3, poly(ADP-ribose) polymerase (PARP) and cleaved PARP were determined using western blot analysis. Following siRNA transfection, ß-catenin and STAT3 protein levels decreased at 72 h. HepG2 cell growth inhibition and early apoptosis in the ß-catenin and STAT3 siRNA co-transfection group were significantly greater than those in the groups transfected with ß-catenin or STAT3 siRNA alone. Decreased caspase-3 and PARP levels, as well as enhanced cleavage of caspase-3 and PARP were observed in the ß-catenin and STAT3 co-transfection group. Simultaneous silencing of ß-catenin and STAT3 using siRNAs resulted in an enhanced loss of cell viability and induction of apoptosis in HepG2 liver cancer cells, suggesting that these genes are promising targets for the further preclinical and clinical development of anti-cancer therapeutic strategies, which target several cancer signaling pathways simultaneously.

An improved method of nasojejunal feeding tube placement for patients requiring endoscopic nasobiliary drainage.

OBJECTIVE: To avoid a second endoscopy for nasojejunal feeding tube placement (NFTP) in patients undergoing endoscopic nasobiliary drainage (ENBD), we studied improved NFTP method and compared it to endoscopic method. METHODS: Patients with ENBD were divided into two groups. One group (18 patients) received endoscopic NFTP and the other group (26 patients) received improved NFTP. Placement time, physical condition of the patients and complications were recorded. RESULTS: In 18 patients who underwent endoscopic NFTP, NFT was successfully placed on the first attempt in 14 patients with a first placement success rate of 77.8%. NFT was wrongly intubated into the trachea in one patient inducing coughing, and after it was removed, the second placement was successful. The total success rate of endoscopic NFTP was 83.3% with an average placement time of 17.0 minutes. In 26 patients undergoing improved NFTP, all were successfully placed on the first attempt with a success rate of 100%, and an average placement time of 2.55 minutes. In patients with ENBD, the success rate of improved NFTP was significantly higher than endoscopic NFTP (χ²=36.4, p<0.05) with a significantly shorter placement time (t=18.5, p<0.05). CONCLUSION: For patients with ENBD, improved NFTP method is superior to the endoscopic method as it is more effective, convenient, faster, and cheaper. Additionally it avoids a second endoscopy and has fewer complications, better security and a higher success rate. The improved method is a safer, easier, more effective and practical method of EN and deserves general adoption in clinical work.

Upregulation of ID2 antagonizes arsenic trioxide-induced antitumor effects in cancer cells.

AIMS AND BACKGROUND: Arsenic trioxide (ATO) strongly induces apoptosis and differentiation in acute promyelocytic leukemia, and induces cell cycle arrest in most solid tumors. Although many signaling pathways are involved in its antitumor mechanism, a detailed investigation of the transforming growth factor beta-bone morphogenetic protein signaling pathway has not been performed. METHODS AND STUDY DESIGN: A microarray containing 113 genes associated with the pathway was used to screen important molecules that participate in the antitumor effects of ATO. The expression levels of the inhibitors of DNA binding-2 (ID2) in 4 different types of cancer cells were determined by quantitative reverse transcription PCR and Western blotting. Human esophageal squamous cell carcinoma cell line Eca109 and pancreatic carcinoma cell line BxPC3 cells were transfected with siRNAs targeting ID2 and scrambled control siRNA. Cell proliferation was evaluated by methyl thiazolyl tetrazolium assay. RESULTS: Eighteen upregulated and 12 downregulated genes were identified. After verification at the transcriptional and translational levels in 4 different cancer cells, ID2 was identified as an ATO antitumor-associated protein. In addition, specific silencing of ID2 could enhance ATO-induced cell proliferation inhibition in cancer cells. CONCLUSIONS: A combination of ATO and ID2-targeted agents may have considerable therapeutic benefits in cancers.